Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are evolutionarily conserved subcellular structures where the ER is closely apposed to the PM. Emerging evidence suggests that ER-PM junctions are important for Ca2+ signaling and lipid metabolism. Nevertheless, the specific functions and regulation of ER-PM junctions remain unclear. We are particularly interested in the role of ER-PM junctions during receptor-induced cell signaling. Receptor stimulation triggers the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) at the PM and the release of Ca2+ in the ER to activate cellular functions including proliferation, migration and secretion. To sustain cell signaling and maintain cell homeostasis, inter-organelle signaling between the ER and the PM is crucial to quickly replenish PM PIP2 and ER Ca2+ with PI in the ER and Ca2+ in the extracellular space, respectively. We have previously identified three proteins, Nir2, E-Syt1 and STIM1, involved in the replenishment of PM PIP2 and ER Ca2+ and localized to ER-PM junctions following receptor stimulation. The objective of this proposal is to investigate the mechanisms of how PIP2 and Ca2+ homeostasis are regulated by dynamic translocation of Nir2, E-Syt1 and STIM1 to ER-PM junctions during receptor-induced cell signaling. We will use molecular, biochemical and genetic approaches in combination with cutting-edge live-cell imaging and EM for the proposed studies. In addition, we will develop innovative tools to monitor signaling events at ER-PM junctions and manipulate selected pools of lipids involved in PIP2 homeostasis. In aim 1, we will determine how PM PIP2 is replenished by dynamic translocation of Nir2 to ER-PM junctions following receptor- induced hydrolysis. In aim 2, we will determine how ER-PM junctions are regulated by dynamic translocation of E-Syt1 following receptor-induced cytosolic Ca2+ increases. In aim 3, we will determine how store-operated Ca2+ entry is regulated by dynamic translocation of STIM1 to ER-PM junctions following ER Ca2+ depletion. We expect the proposed research will establish new paradigms for inter-organelle signaling at membrane junctions and will have a major impact on the fundamental concepts in cell biology and signal transduction.